The NMR structure of the 21 kDa lipocalin FluA, which was previously obtained by combinatorial design, elucidates a reshaped binding site specific for the dye fluorescein resulting from 21 side chain replacements with respect to the parental lipocalin, the naturally occurring bilin-binding protein (BBP). As expected, FluA exhibits the lipocalin fold of BBP, comprising eight antiparallel β-strands forming a β-barrel with an α-helix attached to its side. Comparison of the NMR structure of the free FluA with the X-ray structures of BBP•biliverdin IX γ and FluA•fluorescein complexes revealed significant conformational changes in the binding pocket, which is formed by four loops at the open end of the β-barrel as well as adjoining β-strand segments. An 'induced fit' became apparent for the side-chain conformations of Arg 88 and Phe 99, which contact the bound fluorescein in the complex and undergo concerted rearrangement upon ligand binding. Moreover, slower internal motional modes of the polypeptide backbone were identified by measuring transverse 15 N backbone spin relaxation times in the rotating frame for the free FluA and also the FluA•fluorescein complex. A reduction of such motions was detected upon complex formation, indicating rigidification of the protein structure and loss of conformational entropy. This hypothesis was confirmed by isothermal titration calorimetry, showing that ligand binding is enthalpy driven, thus overcompensating negative entropy associated with both ligand binding per se and rigidification of the protein. Our investigation of the solution structure and dynamics as well as thermodynamics of lipocalin-ligand interaction does not only provide insight into the general mechanism of small molecule accommodation in the deep and narrow cavity of this abundant class of proteins but will also support the future design of corresponding binding proteins with novel specificities, so-called "anticalins".
NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript sequence homology is low (usually only 10-20%), lipocalins share a common fold comprising an eight-stranded antiparallel β-barrel with an attached α-helix (Figure 1). On one end, the β-barrel is "closed" by short loops and densely packed side chains that form the hydrophobic core. On the other end, the β-barrel is usually "open" to the solvent: there, four loops connecting neighboring β-strands form the entrance to the ligand-binding site. Whereas the β-barrel structure is strictly conserved among lipocalins, the loops around the ligand pocket are highly variable regarding length, amino acid sequence and backbone conformation(2). This finding provides a rationale for the diversity of binding specificities observed for this protein family in nature. In this respect, lipocalins may be compared with immunoglobulins, which carry a set of six structurally hypervariable loops on top of a rigid β-sheet sandwich, thus giving rise to a vast repertoire of antigen specificities(3).Based on this notion, the lipocalin scaffold w...
